Abstracts of the 15th Annual Meeting of the ESHRE, Tours, France 1999
P-258. Effect of IGFI and IGFII on PGE2and PGF2a release by human luteal cells
Apa R., Miceli F., Pierro E., Minici F., Fracasso A.M., Tropea A., Mancuso S. and Lanzone A.
Department of Obstetrics and Gynecology, Universita Cattolica S. Cuoro, L.go A. Gemelli 8, Roma, Italy
Introduction: The relevance of the insulin-like growth factor (IGFs) system in ovarian physiology has been extensively demonstrated. Moreover, IGFs have been shown to enhance steroidogenesis both in human granulosa and luteal cells. Taking into account the reports that prostaglandin E2 (PGE2) and prostaglandin F2a(PGF2a ) have opposite (i.e. luteotropic and luteolytic) effects on the human corpus luteum, we aimed to study whether a correlation exists between IGFs effect on progesterone secretion and the activation of the prosta-glandin system.
Materials and methods: Isolated human luteal cells were cultured for different times (12, 24 and 48 h) with IGFI, IGFII (10-100 ng/ml) and GH (100 ng/ml); PGE2and PGF2arelease in culture media was assayed by radioimmunoassay.
Results: Both IGFI and II were able to induce PGE2synthesis in a time-and dose-dependent way while they both inhibited PGF2a production. GH was also found able to significantly reduce PGF2a synthesis; this effect was IGFI-mediated since it was reverted by increasing concentrations of anti-IGFI antibody. Conversely, GH had no effect on PGE2production.
Discussion: In a recent paper of ours, we showed that IGFI as well as II are able to stimulate progesterone secretion by human luteal cells, suggesting a direct luteotropic action exerted by these substances. In light of the evidence of a well-defined correlation between IGFs and prostaglandin release by luteal cells, it is possible to suggest that IGFs could influence luteal steroidogenesis by regulating the complex balance of luteotropic and luteolytic effects exerted by the two different prostanoids.
P-259. Characterization of an immortalized human granulosa cell line (COV434) for the study of the development of ovarian folliclesin vitro*
De Geyter Ch.1,Zhang H.1, Vollmer M.1,De Geyter M.1, Miny P.2 and Holzgreve W.1
1University Women's Hospital of Basel, Switzerland and 2Division of Human Genetics at the Children's Hospital of the University of Basel, Switzerland
Introduction: The biological properties of an immortalized human granulosa cell line (COV434) are presented. The following characteristics were considered essential for normal functionality of granulosa cells: (i) increased synthesis and secretion of 17p-oestradiol after stimulation of cytochrome P450 aromatase with follicle stimulating hormone (FSH); (ii) establishment of intercellular connections between the immortalized granulosa cells and cells of a cumulus oophorus; 270
and (iii) the potential response to similar inducers of apoptosis as compared to natural granulosa cells.
Material and methods: The cell line of immortalized granu-losa cells was established from a primary human granugranu-losa cell tumour. Control granulosa cells were collected from patients undergoing in-vitro fertilization (IVF) or intracyto-plasmic sperm injection (ICSI). The cells were studied under light microscopy , with transmission electron microscopy and with scanning electron microscopy under various conditions. To assess the aromatase activity the cells were cultured in Dulbecco's minimal essential medium (DMEM), supplemented with 10% fetal calf serum (FCS) and 4-androstene-3, 17-dione in presence of recombinant FSH, luteinizing hormone (LH) or urinary human chorionic gonadotrophin (HCG) and the concentrations of 17p-oestradiol and progesterone were meas-ured in the supernatant. mRNA of apoptosis-associated genes were analysed by reverse transcription-polymerase chain reac-tion (RT-PCR) and various apoptotic protein expressions were examined by Western blotting.
Results: Aromatase activity in the COY434-granulosa cells was demonstrated by the rapid rise of the 17p-oestradiol concentration in presence of FSH. No rise of progesterone concentration was observed after the addition LH or HCG neither could the LH-receptor be induced with FSH and l7P-oestradiol. Expression of various apoptosis-associated genes was similar in both granulosa cell types. Multiple intercellular connections were formed during co-culture of COy434-granu-losa cells with cumulus cells containing a viable oocyte but not with cumulus cells devoid of an oocyte. Co-cultured with COy434-granulosa cells, immature human oocytes remained viable for 8 days, but degenerated thereafter.
Conclusions: The immortalized human granulosa cell line COY434 contains many of the properties of natural granulosa cells and may be instrumentalized for experimental studies on follicular development.
*Project nr. 31-49834.96, funded by the Swiss National Foundation.
P-260. Pattern ofcycle-oxygenaseexpression in the human endometrium during the menstrual cycle
Kwon H.C., Yang H.W., Hwang K.J., Kim S.K.l, Cho D.J.1 and Oh K.S.
Department of Obstetrics and Gynaecology, Ajou University School of Medicine, Suwon and 1Department of Obstetrics and Gynaecologyn Yonsei University College of Medicine, Seoul, Korea
Introduction: Recent attention has been focused on the implantation stage endometrial changes at the initiation of decidual reaction induced by prostaglandins (PGs). PGs are derived from arachidonic acid by the action of cyclo-oxygenase (COX), and are comprised of two isoforms, l and COX-2. COX-1 is a constitutional enzyme, and is present in many human tissues, whereas COX-2 is induced by endocrinological stimulation. The expression of COX-l is relatively uniform in the oestrus cycle of mice, but the expression of COX-2 has
